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Revision as of 22:15, 20 October 2007

Proposal for a BioBrick extension for fusion proteins

iGEM team Freiburg

Introduction

Proposal

iGEM parts for mix-and-match construction of fusion proteins

proposed by Katja M. Arndt, Kristian M. Müller, University of Freiburg

• Both parts are compatible with all standard iGEM parts as they have the BioBricks prefix for coding sequences and the standard BioBrick suffix.
• nnnnnn is a place holder for the coding sequence of the respective part.
• Both parts have two additional enzymes, NgoMIV and AgeI, which have compatible cohesive ends and enable in-frame fusion of protein parts with the linker sequence TG (no stop codons).
• The only difference of the N-part and FusionPart is the additional NgoMIV site in the FusionPart.
• The N-part is designed to be the start of a fusion protein or a stand-alone protein part, to be cloned via XbaI/PstI to any iGEM RBS expression part.
• The FusionPart is designed to be fused to the N-part by digesting the N-part with AgeI/SpeI and the FusionPart with NgoMIV/SpeI.
• Any number of FusionParts can be combined and fused to the N-part.
• The FusionPart can also be a stand-alone part as it has a start codon after the XbaI site (BioBrick prefix for coding sequence), however two additional amino acids (A, G) are encoded before the start of the protein.

N-part

                             BsrFI                    SfcI
 ApoI                        BsaWI                 MspA1I|
EcoRI   NotI    XbaI          AgeI      SpeI    NotI    ||PstI
    |      |       |             |         |       |    ||   |
    GAATTCgcggccgctTCTAGAtgnnnnnnACCGGTtaatACTAGTagcggccgCTGCAG
  1 ---------+---------+---------+---------+---------+--------- 59
    CTTAAGcgccggcgaAGATCTacnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC
c     I  R  G  R  F  *  M  ?  ?  T  G  *  Y  *  *  R  P  L  Q   -

FusionPart

                          NaeI     BsrFI                    SfcI
 ApoI                  BsrFI |     BsaWI                 MspA1I|
EcoRI   NotI    XbaI  NgoMIV |      AgeI      SpeI    NotI    ||PstI
    |      |       |       | |         |         |       |    ||   |
    GAATTCgcggccgctTCTAGAtgGCCGGCnnnnnnACCGGTtaatACTAGTagcggccgCTGCAG
  1 ---------+---------+---------+---------+---------+---------+----- 65
    CTTAAGcgccggcgaAGATCTacCGGCCGnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC
c     I  R  G  R  F  *  M  A  G  ?  ?  T  G  *  Y  *  *  R  P  L  Q   -

Discussion

References